Trioecy is maintained as a time-stable mating system in the pink sea urchin Toxopneustes roseus from the Mexican Pacific

Trioecy is a sexual system that consists of the co-occurrence of females, males and hermaphrodites in a population and is common in plants; however, in animals it is uncommon and poorly understood. In echinoderms, trioecy had never been recorded until now. Frequencies of females, males, and hermaphrodites were evaluated and gametogenic development was histologically characterized in a population of Toxopneustes roseus inhabiting the Mexican Pacific. Trioecy in this population is functional and temporally stable, since the three sexes coexisted in each sampling month. The hermaphrodites presented similar gametogenic development as the females and males and participated during the spawning season, contributing to the population’s reproductive process. Trioecy is considered an evolutionarily transitory state, and it is extremely difficult to explain its presence in a species. We hypothesize that continuous ocean warming represents a threat to the survival of this population of T. roseus, since its early developmental stages, which represent a population bottleneck, are more vulnerable to high temperatures than other sea urchins inhabiting the area, while its population density is significantly lower. These conditions generate a strongly stressed environment, which is the determining factor that maintains the stability of trioecy in the species in which it has been studied.

Trioecy is a sexual system that consists of the co-occurrence of females, males and hermaphrodites in a population and is common in plants; however, in animals it is uncommon and poorly understood. In echinoderms, trioecy had never been recorded until now. Frequencies of females, males, and hermaphrodites were evaluated and gametogenic development was histologically characterized in a population of Toxopneustes roseus inhabiting the Mexican Pacific. Trioecy in this population is functional and temporally stable, since the three sexes coexisted in each sampling month. The hermaphrodites presented similar gametogenic development as the females and males and participated during the spawning season, contributing to the population's reproductive process. Trioecy is considered an evolutionarily transitory state, and it is extremely difficult to explain its presence in a species. We hypothesize that continuous ocean warming represents a threat to the survival of this population of T. roseus, since its early developmental stages, which represent a population bottleneck, are more vulnerable to high temperatures than other sea urchins inhabiting the area, while its population density is significantly lower. These conditions generate a strongly stressed environment, which is the determining factor that maintains the stability of trioecy in the species in which it has been studied.
Reproduction and the way in which any living organism reproduces is the fundamental objective in its life history and important to ensure its permanence in ecosystems. Therefore, the biological success of any species involves its members remaining alive long enough to be able to reproduce 1,2 . In nature, there are two types of reproduction: sexual and asexual. Sexual reproduction consists of a complex and sophisticated mechanism, which involves the recombination of an individual´s own hereditary material and the exchange or fusion with the hereditary material of another individual. This type of reproduction increases genetic variability in offspring by producing unique combinations of genes inherited by parents, which makes possible the continuous biological evolution of living beings and their adaptation to the environment 3 .
Whereas different breeding systems exist amongst animals 4 , there are two common sexual systems, gonochorism (or dioecy), in which individuals are either female or male during their reproductive lives but not both, and hermaphroditism, in which the individual produces both male and female gametes during their breeding lives 5 . Most animal species are gonochoric with hermaphroditism occurring in a very low proportion (5-6%) of species 6 . In addition, there are rare mixed reproductive systems, including androdioecy (males and hermaphrodites), gynodioecy (females and hermaphrodites), and trioecy (co-occurrence of males, females, and hermaphrodites).
The condition of the coexistence of males, females, and hermaphrodites is a rare mating system that had previously been documented only in plants, and according to the theory of sex assignment, it should not occur in metazoans within discrete generations and considering simple theory 7 . However, some studies have recently shown that there is evidence of trioecy in two species of microscopic nematodes of the new genus Auanema (A. rhodensis, aka Rhabditis sp. SB347 and A. freiburgensis, aka Rhabditis sp. SB372), and three species of the

Material and methods
Field work. Between  , which is a semi-protected bay surrounded by cliffs. The bay has a thick sandy substrate, a moderate slope and a depth of approximately 12 m (Fig. 1). The bottom has uniform extensions of coral reefs parallel to the coastline and sandy extensions with fragments of dead coral.
All the sea urchins collected (n = 224) had more than 50 mm of test diameter; therefore, they were adults that had already reached the size of sexual maturity 38  Based on the analysis of the histological sections, the sex of each specimen was determined as well as the reproductive stage in which it was found. The followed criteria consisted of the distinctive cellular characteristics of the nucleus, cytoplasm, and follicular and germinal epithelium walls, as well as the distribution and size of the gametes in the gonadal follicles 39,40 . For the specific case of oocytes, the nucleus-cytoplasm relationship was also considered, as well as their affinity for the dyes.
Welfare, humanitarian sacrifice, and ethically responsible research with the sea urchins were in accordance with the ethical recommendations for humanitarian killing of animals as established under Mexican law (NOM-033-SAG/ZOO-2014). No live vertebrates were handled during this study. Data analysis. The sexual proportion of the specimens was calculated considering males, females and hermaphrodites. The proportions were analyzed using the Chi square test (X 2 ) to determine if there was a significant deviation from a 1F: 1 M: 1Hp (Female: Male: Hermaphrodite) ratio. The decision rule was made with a 95% confidence interval, rejecting a 1: 1: 1 ratio when the calculated X 2 value was greater than 5.99 41,42 .
Of the 32 specimens with the presence of gametes of both sexes inside or outside the follicles, 27 (84.3%) had a higher proportion of male gonadal tissue (Fig. 3A), while three of the individuals (9.3%) had a higher presence of female gonadal tissue (Fig. 3B). Only two individuals (6.2%) presented the same proportion of tissue of both sexes (Fig. 3C).
When analyzing the phases of gonadal development by sex, in females the resting phase occurred in three months (April 2015, August 2015, and January 2016), and the highest frequency of individuals in this phase occurred in August. The growth phase occurred almost every month, except in April, May, September, and In males, the resting phase did not appear. The growth phase was observed over four months, with the highest frequency in January 2016. The prematurity phase occurred synchronously with that of the females. The maturity phase began in April and ended in December 2015, and the highest frequencies of individuals in this stage were observed in May and September. Spawning occurred from July to December with the highest frequencies in August and October 2015 (Fig. 4B).
In hermaphrodites, the resting phase did not appear. The growth phase was observed in four months. The prematurity phase was only observed in February 2016 with a frequency of 50%. Maturity was recorded in the first three months with a frequency of 100%. The individuals began to spawn in August, increased in September, and all were spawning in December 2015 and again in March 2016 (Fig. 4C).
Histologically, the gonadal development stages of hermaphrodites were similar to those of dioecious specimens. The ovarian proportions possessed all cell types showing normal progression of oocyte formation from oogonia.
In the growth stage, the presence of spermatogonia was observed in the periphery of the follicular wall in the gonads that contained developing male gametes. This is where spermatogenesis began heading towards the center, which concentrated the spermatozoa in the lumen of the follicle. The gametes were dark purple in color. In some follicles the presence of previthellogenic oocytes was observed, of which some were located near the periphery of the wall and others in the center of the lumen (Fig. 5A). In follicles containing female gametes, the presence of oocytes at different stages of development was observed. Previtellogenic and vitellogenic oocytes as well as nutritive phagocytes were observed in the lumen. The gonadal wall was thick, and previtellogenic oocytes and oogonia were observed near the periphery of the follicle (Fig. 5B). www.nature.com/scientificreports/ In maturing hermaphrodites, follicles that only contained sperm could be identified, while in others there were only oocytes. The follicles containing only spermatozoa were characterized by presenting mature spermatozoa in the lumen and a band of primary sex cells was observed near the wall of the follicle (Fig. 5C). In the case of follicles with oocytes, these were observed at different stages of development, from oogonia to mature oocytes. The presence of a well-defined nucleolus was observed in previtellogenic oocytes, and most of these were found in the periphery of the follicle (Fig. 5C).
The individuals that presented follicles that were releasing spermatozoa were characterized by presenting empty areas between spermatozoa, and in some cases in which the release was partial, the gonadal wall was thin. Likewise, in some follicles the release of oocytes could be observed, which was characterized by presenting spaces that had previously been occupied by mature oocytes in the lumen, and the presence of remaining oocytes was also recorded (Fig. 5D).

Discussion
According to the information that exists so far regarding reproduction in echinoderms, this is the first work in which the occurrence of trioecy in sea urchins is reported. This is also the first report of trioecy among members of the phylum Echinodermata, one of the most widespread taxa, both latitudinally and bathymetrically. Our results show that trioecy in this population of T. roseus is temporally stable, since the three sexes were observed together throughout the year in each month of sampling. Hermaphroditic individuals also presented the same gametogenic developmental pattern as females and males. Finally, during the spawning period of the population they contributed to the reproductive process by releasing mature gametes, which evidenced their full functionality within the studied population.
We were unable to obtain evidence of self-fertilization in the studied hermaphrodites; but self-fertilization in the gonads and gonadal ducts of a hermaphrodite individual of Echinocardium cordatum was recorded in 1935 43 . However, the embryos produced did not complete development successfully, probably due to the premature fertilization within the gonad 43 . Also, the cases of fully functional hermaphrodites of Arbacia punctulata have been reported 44,45 . The gametes of the hermaphrodites were fertilized as soon as they were released into seawater and the development of self-fertilized eggs was absolutely normal in time and morphology. After nine days, typical pluteus larvae were obtained and both the eggs and sperm of the hermaphrodites functioned ordinarily with gametes from other males and females.
Therefore, we consider that there are no reasons to think that in the case of Toxopneustes roseus hermaphrodites cannot carry out self-fertilization. According to the analysis of the gonad developmental stages, their gametes were released into seawater. Theoretically, those gametes would be able to follow the normal course of fertilization, interacting among them and with gametes of females and males. www.nature.com/scientificreports/ The trioecic condition has been recorded so far only in some animals, such as a few nematode species and a hydra 9,10,14,46-48 . In marine invertebrates, it has been reported in one anemone under laboratory conditions and in one bivalve mollusk 15,16 . The coexistence of males, females and hermaphrodites has been considered an evolutionarily transitory state; for example, androdioecy (male / hermaphrodite) in nematodes such as Caenorhabditis elegans is believed to have evolved from dioecy (male / female) through a trioic intermediate. Consequently, it is very difficult to find the ecological or evolutionary causes that lead a species or population to present three sexes simultaneously 49 .
In the species in which trioecy has been studied and monitored, it is noticeable that their populations are subjected to strong environmental stress in situ or under laboratory manipulation [50][51][52] . For example, some nematodes of the genus Tokorhabditis are extremophilic species that live in the Californian Mono Lake, which is characterized by being hypersaline and exhibiting high levels of arsenic 10,50 . In the case of Auanema freiburgensis the flexible sex determination and mating system and, consequently, its trioecy can be critical for resilience at the population level in patchy, resource-limited environments 49 . These results thus demonstrate that life-history, ecology and environment can play defining roles in the development of sexual systems and determine the continued presence of trioecy in the nematode. In the case of Hydra viridissima, it unlike most European species, is a "warm crisis" hydra, since it usually reproduces asexually, but when the temperatures rise to, or are maintained at high levels (≥ 20 °C), it reproduces sexually 14,53 . In experimental conditions, the population studied essentially behaved as androdioecic and only at the end of the research period, when the temperature was the highest (~ 25 °C), a few females appeared and joined the other existing sexes, thus generating the condition of trioecy 14 . Trioecy has been identified in another non-described species (e.g., Rhabditis sp. JU1783) isolated from star fruit, although it is closely related to A. rhodensis and A. freiburgensis and likely to belong to the same genus 11,12 . Little is known about the ecology of Auanema, as A. rhodensis has been isolated from a tick and a beetle, and A. freiburgensis from dung and a rotting plant of the genus Petasites 12,47,51 .
Regarding the sea anemone Aiptasia diaphana, it is mainly found in isolated fouling communities, and no hermaphrodites exist in natural populations that could reproduce asexually or sexually 54 . However, under laboratory conditions, a single founder individual (asexual clone) produced not only males and females, but also www.nature.com/scientificreports/ hermaphroditic individuals. In addition, A. diaphana can fertilize within and between cloning lines, producing larval-swimming planules, which could explain the success of the species as an invader of artificial marine substrates. The condition of trioecy was also identified in individuals of this anemone manipulated in the laboratory, to create age-homogeneous populations of asexual propagules (pedal lacerations) and ontogenetic patterns of sexual differentiation were documented 15 .
In the case of the marine bivalve Semimytilus algosus, there was not an obvious explanation for the occurrence of its trioecy, despite the intense analyses of factors such as motility versus a sessile way of life or reproductive density within a population, which could have relevance for gamete interactions 16 . In many respects, S. algosus is a "typical" marine intertidal mussel, since it is sessile in adulthood, occurs at high densities in wild populations, and has a very large population. S. algosus also co-occurs with other species that are close relatives within the Mytilidae family and have evolved and conserved their dioecy 16 .
Toxopneustes roseus is another typical species of sea urchin, which has a wide latitudinal distribution throughout the tropical eastern Pacific and co-inhabits with other species of sea urchins and echinoderms that have a similar distribution and in which hermaphroditism has not been reported 40,[55][56][57] . Regarding its population density, T. roseus is not considered among the most abundant species in the study area and its densities are relatively low (between 0.04 and 1.2 ind.m 2 ). However, it cannot be considered a rare species in terms of abundance 58,59 .
All of the above makes it difficult to clearly explain the reasons for the occurrence of trioecy in this species; however, certain aspects of its early development are known that could indicate the factors behind the development of this reproductive mating system in the pink sea urchin. In recent experiments carried out with gametes, larvae, and embryos of a population of T. roseus from the same area as our study, it was found that the increase in temperature above the normal values of its habitat has a deleterious effect on the success of early development 60 . There exists experimental evidence that at an increase of temperature to 32 °C, which is 2 °C above the maximum values registered in the study area, fertilization occurred at a very low percentage. There was also a deleterious effect on embryos, resulting in abnormal development and the lowest percentage of larval survival also occurred at 32 °C 60 . The same kind of experiments has been performed on other species from the study area, such as the irregular sea urchin Ryncholampas pacificus and the intertidal Echinometra vanbrunti. The deleterious effects on these species were observed only at 34 °C, which was the highest temperature tested (unpublished data). At 32 °C, however, there was no evidence of negative effects in the case on E. vanbrunti, and there was just arrested development, but no abnormalities in the case of R. pacificus. These results indicate that T. roseus is much more sensitive to the rise in temperature than other cohabiting sea urchins, and probably lives near its upper thermal limit. In that context, the continuous ocean warming could threaten the permanence of the species in the study www.nature.com/scientificreports/ area, since the early stages of development constitute a bottleneck for successful recruitment and later population maintenance in populations that carry out reproduction by means of external fertilization. Within the phylum Echinodermata, when stressful conditions appear in the habitat or the environment becomes hostile, the species can generally resort to asexual reproduction by fission (ophiuroids) or fission and autotomy (holothuroids and asteroids) to increase the abundance of populations in a relatively short time or counteract a threat with numbers 61 . This does not apply to sea urchins since they are unable to reproduce asexually. The only way for sea urchins to reproduce asexually would be by cloning larvae, but this process would also require that sexual reproduction occurs first 62 . Therefore, any reproductive strategy that a sea urchin population could develop to respond to drastic changes in their area must involve sexual reproduction. In this regard, in an experimental evolution study with the nematode Caenorhabditis elegans, in which partial selfing, exclusive selfing, and predominant outcrossing were compared, it was evidenced that monoecious populations only have hermaphrodites and, therefore, reproduction is carried out exclusively by self-fertilization. However, in trioic populations that have males, females, and a small number of hermaphrodites, reproduction is predominantly carried out by external crossing 49 . Also populations that underwent some degree of interbreeding during the evolutionary experiments (trioic and androdioic populations), maintained more genetic diversity than expected solely under genetic drift or under genetic drift and directional selection 49 . In this sense, it is possible that high levels of interbreeding, such as that which occurs in trioic populations, develop with populations that have sufficient deleterious recessive alleles to avoid extinction, since selection is less efficient to purge them. Trioecy, therefore, becomes an efficient system to select characteristics of the genome that allows a population that only reproduces sexually to adequately cope with significant changes in the environment that could threaten the permanence of the species in that habitat. Interbreeding (gonochorism, self-incompatible hermaphroditism) also favors genetic diversity and offers greater potential to adapt to changing environments 63 . The costs and advantages of crossing over selfing depend on environmental factors and, therefore, selection may favor transitions between mating systems. Androdioecy, gynodioecy, and trioecy are evolutionarily unstable intermediate strategies, but they offer important systems for testing models of the causes and consequences of the mating system in the evolution of populations 63 .
However, the question remains why T. roseus has developed trioecy, when in the same habitat there are other sea urchins with very similar life-histories that only maintain dioecy. In the case of the bivalve Semimytilus algosus; which presents the same situation as we have with T. roseus, it was proposed that the trioecy of the species may be related to the sex determination mechanism, considering what it is known about the nematodes of the genus Auanema 10,16,46 . In Auanema, the male versus non-male (hermaphrodite or female) decision is determined genetically (XO for males, and XX for females and hermaphrodites) 9,64 . The hermaphrodite versus female decision, however, is determined by the environment of the mother. For A. freiburgensis the maternal social environment is determinant, whereas for A. rhodensis it is the age of the mother 9,12,51,65 . Therefore, in Auanema, environmental sex determination and genetic sex determination interact to produce trioecy.
Although there is apparently no clear cause of strong, stressful conditions in the habitat of T. roseus that could threaten the survival of this species, according to the United States Environmental Protection Agency (EPA, 2021), sea surface temperature increased during the twentieth century and continues to rise. From 1901 to 2020, the global temperature rose at an average rate of 0.004 °C per decade, resulting in a total increase of 0.5 °C to date. Additionally, regional studies based on continuous monitoring, which have not yet been published, have shown that between 2002 and 2020 there has been an increase of approximately 1 °C above the historical average of the sea surface temperature in the study area.
The foregoing discussion leads us to speculate that the studied population of T. roseus lives at the limit of its thermal tolerance, and the constant increase in ocean temperature due to global warming constitutes a threat to its survival and a constant source of stress for the population. This is because its early-development stages are more vulnerable to high temperature than other sea urchins that live in the same area and its population density is also significantly lower 58 .
Phylogenetically T. roseus belongs to Family Toxopneustidae and although no other species within the genus Toxopneustes has shown hermaphroditism, this condition was reported in Tripneustes gratilla, which belongs to the same family 36 . Toxopneustids belong to the Order Camarodonta, and almost all the species of sea urchins in which hermaphroditism has been reported belong to this Order except for a couple that belong to the Arbacioida. At the same time, this order is contained in the Superorder Echinacea along with Camarodonta, according to the last exhaustive analysis resolving the position of the clades within Echinoidea 66 . In this context, theoretically T. roseus at some point underwent the environmental pressure of its early stage living under constantly rising temperatures, along with its low population densities in the study area. Consequently, it was able to develop hermaphroditism and, therefore, trioecy, similarly to what occurred to Hydra viridissima under conditions of extreme high temperature 14 . We hypothesize that these permanent conditions generate a constant source of strong environmental stress, which is the determining factor that keeps trioecy stable in the species in which it has been studied, and, thus, trioecy remains stable in this population of T. roseus.
The mechanism of sex determination in echinoids, as well as in other echinoderms, is still unknown, although the sex ratio, which is generally close to 1:1, suggests that it occurs through sex chromosomes 67 . It is known that in mammals, sex determination is dictated by the presence or absence of the Y-chromosomal gene SRY. SRY functions as the primary sex-determining gene by activating testis formation, and in its absence, the embryo will form ovaries. SRY only exists in mammals; however it evolved as a duplication of the Sox gene family, which exists in all metazoans 68 .
In vertebrates, Sox genes are involved in sex determination, neurogenesis, skeletonogenesis, eye development, pituitary development, pancreas formation, and neural crest and notochord formation 69 . In invertebrates, they are involved in processes such as metamorphosis, eye development, neural crest formation, and ectoderm formation 70  www.nature.com/scientificreports/ primordial gut during development and is closely related in sequence to Sox genes of the mouse embryo 71 . An investigation of sex determination was carried out in the sea urchin Strongylocentrotus purpuratus using RNA-seq and quantitative mRNA measurements, but the mechanisms that govern sexual determination of the species could not be clearly established 72 . However; the results show that the male fate factors Dmrt and SoxH are expressed early and meiosis initiates early. Also, gonad-specific transcripts involved in egg and sperm biology, are first activated before rudiment formation in the larvae of this sea urchin. The study provided additional evidence for the hypothesis that in sea urchins, sex determination occurs genetically 72 . Another research with the sea cucumber Apostichopus japonicus, which integrated genome-wide association study and analyzes of sexspecific variations evidenced that the species exhibits genetic sexual determination 73 . Furthermore, analysis of homozygous and heterozygous genotypes of abundant sex-specific SNPs in females and males, confirmed that A.japonicus might have a XX/XY sex determination system 73 .
On the other hand, it has been proposed that a deviation from the 1:1 sex ratio in echinoids could reflect environmental conditions that influence sex determination 67 . For example, a relatively large proportion of Lytechinus variegatus and Tripneustes ventricosus (as Tripneustes esculentus) hermaphrodites was recorded in southern Florida during an unusually cold winter, suggesting that adverse winter conditions in some way affected sex determination in juveniles 74,75 . Also relatively large number of Strongylocentrotus purpuratus hermaphrodites was reported in Bahía de Todos los Santos, Mexico, where extreme seasonal fluctuations in temperature (from about 12-24 °C) are recorded 76 . However, posterior studies did not find a single hermaphrodite of Strongylocentrotus purpuratus in more than 500 individuals analyzed 77,78 .
Considering that sex determination in sea urchins is highly probable to occur genetically and the possibility that the environment may also influence sex determination, we think that in the case of Toxopneustes roseus, genetic sex determination and environmental sex determination are interacting to maintain the condition of trioecy stable. We propose that, especially because the cases in which environmental conditions have assumed to influence sex determination, extreme temperatures are invoked as the main affecting factor. However, more detailed studies are needed in terms of sexual determination and experimental evolution to be able to verify our assumption.
In general, the efforts that have been made to explain the evolution of the sexes and the origin of hermaphroditism and trioecy are still scarce, and critical questions remain to be answered. The case of trioecy detected in T. roseus may constitute an important model to seek these answers about the evolution of sexual systems and the environmental mechanisms that trigger trioecy in marine macroinvertebrates and, in particular, in echinoderms.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/